The present invention relates to a device for detecting microbial growth and enumerating the microorganisms present in a sample, and means to accelerate their growth rate.
It is necessary to test various industrial samples, such as food, pharmaceuticals, cosmetics and water for microbial contamination One area of microbiological testing involves the estimation of total number of bacteria, yeast and molds as well as contamination of specific groups of organisms in the sample. One widely used method is known as the xe2x80x9cStandard Plate Countxe2x80x9d method and involves culturing a diluted sample in an agar growth medium The plates containing the sample and the growth medium are incubated (e.g., 32xc2x0 C.-40xc2x0 C.) for 24 hours to 5 days depending upon the assay. After incubation, colonies of microorganisms that have been grown upon the agar surface are counted. Optical methods based upon color change have been successfully used to classify microorganisms in clinical samples. Turner describes in U.S. Pat. No. 4,945,060 noninvasive means for detecting the presence of microorganisms in clinical specimens with an optical sensor located at the bottom of a culture bottle. The sensor comprises a solid composition or a membrane with an indicator substance immobilized on or within it. The metabolic products of the organisms growing in the liquid media diffusse to the solid sensor and change the color of the indicator. Similar devices are disclosed in U.S. Pat. Nos. 5,094,955, 5,164,796, 5,217,876 and PCT WO 96/39533. All of these devices comprise sensors made of a semi-permeable layer such as a membrane or cured polymer, The problem with these devices is that the diffusion rate of the metabolic products is not uniform and can vary from one culture bottle to another. Consequently these devices have been used to detect only the presence of microorganisms, and not for enumeration tests.
Another device, disclosed by Eden in U.S. Pat. No. 5,366,873, comprises a container with growth media and indicator substance disposed in the media for undergoing transformation in the presence of organisms growth. Since the indicator substance is in the media, changes in color due to microbial growth are detected rapidly and consistently in the indicator. A layer of semi-fluid substance, such as agar, containing the same chemical compounds present in the liquid media, is in chemical equilibrium with the media and consequently reflects the changes occurring in the media while preventing the food particles and microorganisms from masking these changes. This device can be used for enumeration of organisms in a sample based upon the consistency of the patterns obtained when using a photometer recording the optical changes of the indicator on-line. The present invention introduces new and different means to change the properties of an optical indicator. The changes result from the generation of gases from metabolic activity of microorganisms.
One of the advantages of the present invention is the capability to detect organisms (e.g. molds) which the devices recited in the prior art cannot detect. Another advantage of the invention is its capability to enhance growth of aerobic microorganisms by employing a highly porous material or an agar slant which increases the surface area for microbial exposure to oxygenated growth media. The concept of the porous material has been introduced in U.S. Pat. No. 5,672,484 and the agar slant is a well-known technology in microbiology. These enhancements are now combined in the new invention in order to optimize the optically based technology disclosed.
In accordance with the present invention an external container with at least one transparent portion contains an indicator capable of changing color due to gases produced by microbial metabolism. An internal container is placed inside the external container and occupies a portion of the headspace of the external container. The internal container contains appropriate growth media to promote microbial growth and the sample to be tested. The uncovered internal container is placed inside the external container and the external container is capped. The assembly is than incubated at an appropriate temperature. Due to microbial growth, gases such as CO2 are produced. The generated gases are released from the internal container to the headspace of the external container and a chemical reaction occurs between the gases and the chemicals in the transparent portion of the external container, causing the indicator to change color. This color change is monitored either visually or optically by an external photometer.